The objective of this research program is to obtain basic information leading to an improved understanding of enzymatically catalyzed glycosylation, and of the relationship of glycoside hydrolases to glycosyl transferases. The glycosylation process effected by these enzymes is of central bilogical importance as the means whereby the varied complex saccharides of living forms are synthesized from (and broken down to) smaller sugar components. Our goal of advancing understanding of the catalytic capabilities of this wide range of enzymes will be approached using a new and highly effective method: the study of productive glycosylation reactions obtained with glycosyl (but nonglycosidic) substrates. Such reactions will be used to probe the catalytic abilities and mechanisms of a variety of purified enzymes that have been considered well characterized on the basis of their actions on glycosidic substrates. Reactions with glycosyl fluorides as substrates will be used to elucidate newly detected catalytic capabilities of pullulanase and trehalase (each previously held to be a strict hydrolase); to investigate the scope of the recently discovered role of acceptor substrates in determining reaction product configuration; to gain insight into the actions of certain active-site-directed inhibitors by their effects on reactions catalyzed by glucoamylase (and by Beta-amylase) with Alpha- and Beta-anomers of the same substrate; to investigate the functional flexibility of the catalytic groups of sucrose, cellobiose and maltose phosphorylases. Newly found reactions with enolic glycosyl donors (the hydration of maltal by Beta-amylase, and of maltotrio-enitol by pullulanase) will be studied to further elucidate the catalytic functioning of these enzymes. In addition, a broad investigation will be made of the catalytic capabilities of various D-glucosyl mobilizing enzymes, using a new type of prochiral C8-enolic glycosyl donor that will allow determination of the full stereochemical course of catalyzed reactions. Collectively, these specific proposals are expected to provide a deeper understanding of the functioning of the catalytic groups of glycosylases in general.